Cell-mediated (type-1) immunity is necessary for immune protection against most intracellular pathogens and, when excessive, can mediate organ-specific autoimmune destruction. Mice deficient in Eta-1 (also called osteopontin) gene expression have severely impaired type-1 immunity to viral infection [herpes simplex virus-type 1 (KOS strain)] and bacterial infection (Listeria monocytogenes) and do not develop sarcoid-type granulomas. Interleukin-12 (IL-12) and interferon-gamma production is diminished, and IL-10 production is increased. A phosphorylation-dependent interaction between the amino-terminal portion of Eta-1 and its integrin receptor stimulated IL-12 expression, whereas a phosphorylation-independent interaction with CD44 inhibited IL-10 expression. These findings identify Eta-1 as a key cytokine that sets the stage for efficient type-1 immune responses through differential regulation of macrophage IL-12 and IL-10 cytokine expression.

Osteopontin (OPN) is a phosphorylated acidic glycoprotein that has been implicated in a number of physiological and pathological events, including maintenance or reconfiguration of tissue integrity during inflammatory processes. As such, it is required for stress-induced bone remodeling and certain types of cell-mediated immunity. It also acts in dystrophic calcification, coronary restenosis, and tumor cell metastasis. An RGD-containing protein, OPN exists both as an immobilized ECM molecule in mineralized tissues and as a cytokine in body fluids; it is not a significant part of typical nonmineralized ECM.OPN can engage a number of receptors, including the integrins α v (β 1 , β 3 , or β 5 ) and (α 4 , α 5 , α 8 , or α 9 )β 1 , and it may also be a ligand for certain variant forms of CD44, specifically v6 and/or v7, but possibly only in conjunction with a β 1 integrin (1). These receptors directly or indirectly activate cellular signaling pathways, allowing OPN to mediate cell-matrix, and possibly cell-cell, interactions. Several studies have demonstrated that OPN delivers a prosurvival, antiapoptotic signal to the cell. Here, we argue that OPN influences cellular functions in a unique manner, by mimicking key aspects of an ECM signal outside the confines of the ECM. We will explore this idea by reviewing recent data concerning OPN signaling and the consequences of OPN deficiency in several settings, notably inflammatory processes involving immune cells and bone cells. Figure 1 illustrates some of the features of the OPN molecule. The presence of a conserved thrombin cleavage site suggests that certain physiological processes employing OPN depend upon its cleavage by thrombin. Some of these adhesive interactions involve the RGD sequence, which is found in various ECM proteins and binds directly to many integrins. Both RGD-dependent and RGD-independent OPN-receptor interactions are modulated by thrombin cleavage of OPN. For instance, thrombin-cleaved OPN, but not intact OPN, can support RGD-dependent migration of melanoma cells (2). Likewise, K562 erythroleukemia cells bind via activated α 5 β 1 to the RGD sequence in thrombin-cleaved OPN. A non-RGD-dependent interaction with α 9 β 1 offers yet another example: only after cleavage by thrombin can human OPN interact with α 9 β 1 via the sequence SVVYGLR, which is located between the RGD sequence and the thrombin cleavage site (3). This binding motif is also responsible for the RGD-independent binding of the J6 T-cell line to activated α 4 β 1 , but in the latter case, cleavage by thrombin is not required for binding of OPN by activated integrin (4). Adhesion of B lymphocytes via α v β 3 also occurs via a cryptic binding site masked in intact OPN, and TPA-activated B lymphocytes attach more effectively to thrombincleaved OPN than to full-length OPN (5). In contrast, Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival OPN-integrin interactions: consequences of cleavage by thrombin

In this review most of the various known, suspected, or postulated functions of osteopontin, a secreted highly acidic phosphoprotein, are discussed in terms of what we currently know about the protein. These include 1) binding of OPN both to cells via a GRGDS cell adhesion sequence that recognizes the alpha v beta 3 integrin and to extracellular matrix components via poorly characterized motifs, 2) regulation of the formation and remodeling of mineralized tissue, 3) recruiting and stimulating macrophages and lymphocytes as part of a nonspecific response to microbial infections, 4) multiple interactions with Ca2+ that likely influence OPN protein conformation and may be important in Ca(2+)-mediated or Ca(2+)-dependent processes, 5) inhibiting the growth of calcium oxalate crystals by disruption of the growing crystal lattice, 6) effects on gene expression, Ca2+ regulation, and nitric oxide production, and 7) involvement in cell migration. OPN production is frequently augmented when cell signaling pathways are activated by any of a variety of stimuli, for example in cancer cells.

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Although recent data suggests that osteoblasts play a key role within the hematopoietic stem cell (HSC) niche, the mechanisms underpinning this remain to be fully defined. The studies described herein examine the role in hematopoiesis of Osteopontin (Opn), a multidomain, phosphorylated glycoprotein, synthesized by osteoblasts, with well-described roles in cell adhesion, inflammatory responses, angiogenesis, and tumor metastasis. We demonstrate a previously unrecognized IntroductionHematopoietic stem cell (HSC) engraftment is a multistep process, involving homing, transmarrow migration (TMM), and lodgment within a bone marrow (BM) niche. Homing is the specific recruitment of HSCs to the BM and involves the recognition of HSCs by the BM microvascular endothelium and transendothelial cell migration into the hematopoietic space. Lodgment is defined as the selective migration of HSCs to a suitable niche within the extravascular compartment. In comparison to homing, very little is known about molecules that regulate HSC lodgment and, moreover, the retention of HSCs within these distinct anatomical locations.In accord with the stem cell niche model proposed by Schofield, 1 recent studies within our laboratory demonstrate that HSCs actively migrate toward and reside within the endosteal region at the bone and BM interface. 2,3 This concept is supported by studies reported by Calvi et al, 4 Zhang et al, 5 and Arai et al, 6 which highlight the importance of direct contact and interactions between HSCs and osteoblasts at the endosteal surface in the regulation of HSC proliferation. Further evidence that osteoblasts directly regulate hematopoiesis is provided by studies in which conditional ablation of osteoblasts results in significant reduction of marrow hematopoiesis, 7 although the factors responsible for this profound effect remain to be determined. In addition, in vitro evidence indicates that osteoblastic cells can expand HSC numbers 8 and, when cotransplanted with HSCs, can improve engraftment. 9 Collectively, these findings suggest that osteoblasts are a key cell type within the HSC niche and that molecules expressed by these cells may have previously unrecognized roles in regulating hematopoiesis.One molecule that shows high levels of expression in osteoblasts cells lining bone trabeculae is Osteopontin (Opn), 10 an observation that is not unexpected given its well-described role as a key regulator of bone homeostasis. 11 Opn is a multidomain, phosphorylated glycoprotein synthesized by many cell types and involved in many physiologic and pathologic processes, including cell adhesion, 12 angiogenesis, 13 apoptosis, inflammatory responses, and tumor metastasis. 14 Physiologically, phosphorylation, glycosylation, and cleavage of Opn result in molecular mass variants, ranging from 25 to 75 kDa. The different effects that Opn elicit are attributable to its multiple receptors, binding sites, and its various forms. 15 One of the major serine proteases to cleave Opn is thrombin, giving rise to a 24-kDa and a 45-kDa fragm...

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In an earlier report, we used differential cloning to identify genes that might be critical in controlling arterial neointima formation (Giachelli, C., N. Bae, D. Lombardi, M. Majesky, and S. Schwartz. 1991. Biochem. Biophys. . In this study, we sequenced the complete cDNA and conclusively identified one of these genes, 2B7, as rat osteopontin. Using immunochemistry and in situ hybridization, we found that medial smooth muscle cells (SMC) in uninjured arteries contained very low levels of osteopontin protein and mRNA. Injury to either the adult rat aorta or carotid artery using a balloon catheter initiated a qualitatively similar timedependent increase in both osteopontin protein and mRNA in arterial SMC. Expression was transient and highly localized to neointimal SMC during the proliferative and migratory phases of arterial injury, suggesting a possible role for osteopontin in these processes. In vitro, basic fibroblast growth factor (bFGF), transforming growth factor-,@ 8), and angiotensin II (AII), all proteins implicated in the rat arterial injury response, elevated osteopontin expression in confluent vascular SMC. Finally, we found that osteopontin was a novel component of the human atherosclerotic plaque found most strikingly associated with calcified deposits. These data implicate osteopontin as a potentially important mediator of arterial neointima formation as well as dystrophic calcification that often accompanies this process. (J. Clin. Invest. 1993Invest. . 92:1686Invest. -1696

The features of three distinct protein phosphorylation cascades in mammalian cells are becoming clear. These signalling pathways link receptor-mediated events at the cell surface or intracellular perturbations such as DNA damage to changes in cytoskeletal structure, vesicle transport and altered transcription factor activity. The best known pathway, the Ras-->Raf-->MEK-->ERK cascade [where ERK is extracellular-signal-regulated kinase and MEK is mitogen-activated protein (MAP) kinase/ERK kinase], is typically stimulated strongly by mitogens and growth factors. The other two pathways, stimulated primarily by assorted cytokines, hormones and various forms of stress, predominantly utilize p21 proteins of the Rho family (Rho, Rac and CDC42), although Ras can also participate. Diagnostic of each pathway is the MAP kinase component, which is phosphorylated by a unique dual-specificity kinase on both tyrosine and threonine in one of three motifs (Thr-Glu-Tyr, Thr-Phe-Tyr or Thr-Gly-Tyr), depending upon the pathway. In addition to activating one or more protein phosphorylation cascades, the initiating stimulus may also mobilize a variety of other signalling molecules (e.g. protein kinase C isoforms, phospholipid kinases, G-protein alpha and beta gamma subunits, phospholipases, intracellular Ca2+). These various signals impact to a greater or lesser extent on multiple downstream effectors. Important concepts are that signal transmission often entails the targeted relocation of specific proteins in the cell, and the reversible formation of protein complexes by means of regulated protein phosphorylation. The signalling circuits may be completed by the phosphorylation of upstream effectors by downstream kinases, resulting in a modulation of the signal. Signalling is terminated and the components returned to the ground state largely by dephosphorylation. There is an indeterminant amount of cross-talk among the pathways, and many of the proteins in the pathways belong to families of closely related proteins. The potential for more than one signal to be conveyed down a pathway simultaneously (multiplex signalling) is discussed. The net effect of a given stimulus on the cell is the result of a complex intracellular integration of the intensity and duration of activation of the individual pathways. The specific outcome depends on the particular signalling molecules expressed by the target cells and on the dynamic balance among the pathways.